The body temperature of marine teleosts living in polar waters is in temperature equilibrium with the surrounding sea, and is thus at a temperature of approximately −1.8° C. during winter or year round in for example Antarctic waters. The blood of marine teleosts is hypoosmotic to the seawater and its melting point is predicted to be approximately −0.7° C. The polar teleosts are thus supercooled and lethal freezing would be expected in the absence of an adaptation to such harsh climatic conditions.
Similar observations are applicable for a variety of cold-adapted terrestrial organisms, including arthropods, plants, fungi and bacteria although many of these organisms are subjected to much lower temperatures than marine fish. Psychrophilic (cold-loving) organisms have successfully adapted to all the permanently cold regions on earth: They can be found in the deep sea, on freezing mountain tops and in the Polar regions even at temperatures as low as −60° C. Despite the lethal conditions, these organisms have overcome key barriers inherent to permanently cold environments. These barriers include: reduced enzyme activity, decreased membrane fluidity, altered transport of nutrients and waste products, decreased rates of transcription, translation and cell division, polypeptide cold-denaturation, inappropriate polypeptide folding and intracellular ice formation.
Research on the mechanisms that allow certain organisms to exist at subzero temperatures has revealed that they rely on at least two strategies: Lowering of the freezing point of water (colligatively by synthesis of low molecular weight substances and non-colligatively via synthesis of unique polypeptides) by either inhibiting ice growth or by giving rise to controlled ice crystal formation. Anti-freeze polypeptides (AFPs) and low molecular weight substances, such as polyalcohols, free amino acids and sugars are believed to be responsible for the former process, while Ice Nucleating polypeptides (INPs) are responsible for the latter.
Anti-freeze polypeptides (AFP—in some publications also known as thermal hysteresis polypeptides, THP, or ice structuring polypeptides, ISP) lower the freezing point of a solution substantially while the predicted melting point is only moderately depressed. This means that whereas the freezing point is lowered dramatically, the melting point of the solution is predicted by the colligative melting point depression. This is true for solutions where ice is present—the question as to whether anti-freeze polypeptides can lower the supercooling point of an ice-free solution is largely unsolved.
The displacement of the freezing temperature is limited and rapid ice growth will take place at a sufficiently low temperature. The separation of the melting and freezing temperature is usually referred to as thermal hysteresis (TH) (Knight et al. 1991, Raymond and DeVries 1977, Wilson 1993), and the temperature of ice growth is referred to as the hysteresis freezing point. The difference between the melting point and the hysteresis freezing point is called the hystersis or the anti-freeze activity. A second functionality of the AFPs is in the frozen state, where they show ice recrystallization inhibition (RI). The AFPs inhibit the formation of large crystals at the expense of small crystals at temperatures above the temperature of recrystrallisation. (Knight et l. 1984, 1995, Knight and Duman 1986, Ramløv et al. 1996).
Mechanism of Inhibition of Ice Formation
The mechanism by which anti-freeze polypeptides inhibit ice growth is still under investigation. AFPs seem all to be amphiphilic. This means that they have one part which is more hydrophobic than the rest of the molecule. Hitherto the explanation for their activity is that their hydrophilic side binds to the ice crystal. However, this view has during the last decade been challenged as when looking at ice/water one can with good reason ask which is per definition most hydrophilic—ice or water. Various evidence for the binding of the AFPs to the ice via their hydrophobic side/domains is emerging, but as the exact mechanism for the binding is not known all evidence so far has been circumstantial.
However, consensus at this point in time is that the anti-freeze polypeptides recognize and bind to various ice surface planes, depending on the type (and isoform) of anti-freeze polypeptide. Ice growth stops where the anti-freeze polypeptides are bound, but continues to a certain extent between the anti-freeze molecules (Raymond and DeVries 1977, DeLuca et al. 1998, Marshall et al. 2004). When the curvature of the ice growing between the anti-freeze polypeptide molecules becomes sufficiently large (or curved), ice growth stops due to the increased surface tension of the curved surfaces (known as the Kelvin effect (Atkins and De Paula 2002, Wilson 1994, 2005, Kristiansen 2005)). It is now not energetically favourable for the water molecules to bind to the curved ice surfaces.
The hysteresis freezing point is thus the temperature where it again becomes energetically favourable for the water molecules to bind to the ice and ice growth continues explosively (Knight et al. 1991, Raymond and DeVries 1977, Wilson 1993).
In most fish anti-freeze solutions, spicular ice growth is seen at the hysteresis freezing point. It is assumed that this is due to the fact that the fish anti-freeze polypeptides bind to the prismplanes on the ice crystals but not to the basal planes. Growth at the hysteresis freezing point is in this case due to binding (addition) of water molecules to the basal planes where growth at the prism planes is still inhibited, thus the ice crystals grow like long spears (spicules).
In solutions containing anti-freeze polypeptides from insects the growth pattern at the hysteresis freezing point is more random and it is suggested that the reason for this is that insect anti-freeze polypeptides also bind to the basal planes (also giving rise to the much higher anti-freeze activity observed in solutions from these animals) and growth thus occurs at spots at any place on the ice crystals once the temperature is low enough (the hysteresis freezing point).
When ice growth is occurring at the hysteresis freezing point in the presence of insect anti-freeze polypeptides the ice growth pattern is cauliflower like in stead of spicular. The thermal hysteresis is thus dependent on at least 2 parameters: 1) the type of anti-freeze polypeptide (dependent on organism, isoform) and 2) the concentration, albeit that the concentration dependency shows saturation (DeVries 1983, Kao et al. 1985, Schrag et al. 1982).
Apparently, there is also a positive correlation between the size of the anti-freeze polypeptide molecules and the amount of hysteresis observed (Kao et al. 1985, Schrag et al. 1982, Wu et al. 2001). Apart from the above mentioned parameters determining the anti-freeze effect it is also substantiated that there is a reciprocal relationship between anti-freeze activity and ice crystal fraction (when the ice crystal is within the hysteresis gap). This effect is most noticeable in the case of insect anti-freeze polypeptides (Zachariassen et al. 2002).
Insect-derived Anti-Freeze Polypeptides
Several AFPs have been found in insects. This is presumably due to the exposure of insects to much lower temperatures than those which fish may encounter (the freezing point of sea water is approximately −1.8° C.).
To date, the structure of insect AFPs has been elucidated for 3 species: Tenebrio molitor, Choristoneura fumiferana and Dendroides canadensis. Two of the three characterized AFPs from insects (from T. molitor and D. canadensis) have many structures in common, presumably because they both come from beetles, whereas C. fumiferana is a moth.
An example of an insect AFP that is very well characterized is the AFP from the beetle Tenebrio molitor. This polypeptide is found in a least 9 isoforms which are all very much alike, but with lengths from 84, 96 to 120 amino acids (Liou et al. 1999). In all the 9 known isoforms a repetitive sequence of 12 amino acids is found: T/A-C-T-X-S-X-X-C-X-X-A-X. This sequence is repeated 6 to 9 times in these AFPs. TmAFP is a right handed beta-helix structure of repetitive amino acid sequences. A highly regular array of threonine residues on the flat beta sheet is thought to interact with water/ice, as the distances between the threonine residues is predicted to fit exactly with the oxygen atom positions in the water molecules of the ice lattice structure. This AFP has an extremely regular structure and a predicted high structural stability provided by a high number for cysteine-cysteine sulphur bridges; every sixth amino acid is a cysteine.
From D. canadensis 13 isoforms with varying length and weight (7.3-12.3 kDa) have been isolated (Andorfer and Duman 2000, Duman et al. 2002). In these AFPs the repeating sequence is the same as is found in T. molitor albeit a 13eth amino acid is sometimes present in some of the repeating sequences ((Duman 1998, Li et al 1998a, 1998b). The cysteines are placed exactly in the same positions as in the AFPs from T. molitor (Li et al. 1998a, 1998b).
The amino acid sequences of the AFPs from C. fumiferana are not homologous to the two other known AFP amino acid sequences from insects. However, a sequence T-X-T is found at every 15th amino acid but only the last threonine is conserved; the first Threonine are in many cases substituted with valine (V), arginine (R) or isoleucine (I). The AFPs from C. fumiferana also contain fewer cysteines than the other two insect AFPs (Doucet et al. 2000). Howvever, all cysteines participate in disulfide bindings (Gauthier et al. 1998). Apparently the AFPs from C. fumiferana contains a hydrophobic core and the polypeptides are stabilized by a network of hydrogen bonds and the disulfide bridges, which all together stabilises the structure (Graether et al. 2000, 2003, Leinala et al 2002).